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Journal of Biomedical Optics

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population
Author(s): Henryk Szmacinski; Vladimir Toshchakov; Joseph R. Lakowicz
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Paper Abstract

Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.

Paper Details

Date Published: 25 April 2014
PDF: 10 pages
J. Biomed. Opt. 19(4) 046017 doi: 10.1117/1.JBO.19.4.046017
Published in: Journal of Biomedical Optics Volume 19, Issue 4
Show Author Affiliations
Henryk Szmacinski, Univ. of Maryland School of Medicine (United States)
Vladimir Toshchakov, Univ. of Maryland School of Medicine (United States)
Joseph R. Lakowicz, Univ. of Maryland School of Medicine (United States)


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